The vector-borne protozoan diseases collectively called leishmaniasis affect ~12 million people in 88 countries worldwide. Cutaneous leishmaniasis (CL), the most common form of leishmaniasis, causes a localized inflammatory skin lesion followed by ulceration and usually resolution. A vigorous inflammatory response occurs at the time of ulceration. Observations of human infection lead to the hypothesis that ulceration and an inflammatory response is necessary for CL healing. Bacterial co-infection and secondary infection are common CL complications, but their effects on disease outcome have not been explored. Inflammasome complexes can form in innate immune cells in response to intracellular danger signals, leading to secretion of pro-inflammatory cytokines IL-1 and IL-18 Three NLR proteins, including NLRP3 and NLRC4, activate inflammasome complexes that include the adaptor protein ASC. Staphylococcus aureus and Pseudomonas aeruginosa are bacteria on human skin that activate NLRP3 and NLRC4, respectively. We hypothesize inflammasomes are activated by bacteria present at the ulceration phase of L. major infection, allowing for control of the parasitic infection. To test this hypothesis, we propose the following specific aims: 1) Determine how bacterial co-infection affects the phenotype and cellular elements of the inflammatory response in cutaneous leishmaniasis. We will co-infect mice with L. major plus S. aureus or P. aeruginosa, and study the effects on the course of bacterial and parasite infection, and the host immune response. Our study will test the hypothesis that bacterial co-infection results in an enhanced inflammatory response and local activation of the inflammasome, which in turn contributes to resolution of L. major infection. 2) Examine the role of the inflammasome on the host response to L. major during bacterial infection. Co- infection studies will be repeated in mice deficient in inflammasome components (caspase-1-/-, ASC-/-, NLRP3-/- , NLRC4-/-) to test the effects of inflammasomes on antimicrobial immunity, and bacterial or parasite clearance. 3) Test whether constitutively inducing inflammasome responses changes the outcome and/or host response of L. major infection. Mice will be infected with WT or transgenic L. major expressing flagellin, a known activator of NLRC4. Thus, we will examine whether constitutive activation of the inflammasome promotes parasite clearance and CL lesion resolution. Understanding the effects of bacterial co-infection and inflammasomes in CL will lay the groundwork for future studies of the interactions between the skin microbiome and innate immunity during parasitic infection. The knowledge gained may contribute to improved approaches to treatment of leishmaniasis. This project will further our understanding of an infectious and immunologic disease and enable us to develop better treatment and prevention strategies for this emerging disease, supporting the mission of the NIAID.